Single gyrA Mutation Research Articles

Evolution of antimicrobial resistance in E. coli biofilm treated with high doses of ciprofloxacin.

The evolution of antimicrobial resistance (AMR) has mainly been studied in planktonic bacteria exposed to sub-inhibitory antimicrobial (AM) concentrations. However, in a number of infections that are treated with AMs the bacteria are located in biofilms where they tolerate high doses of AM. In the present study, we continuously exposed biofilm residing E. coli at body temperature to high ciprofloxacin (CIP) concentrations increasing from 4 to 130 times the minimal inhibitory concentration (MIC), i.e., from 0.06 to 2.0 mg/L. After 1 week, the biofilms were full of CIP resistant bacteria. The evolutionary trajectory observed was the same as described in the literature for planktonic bacteria, i.e., starting with a single mutation in the target gene gyrA followed by mutations in parC, gyrB, and parE, as well as in genes for regulation of multidrug efflux pump systems and outer membrane porins. Strains with higher numbers of these mutations also displayed higher MIC values. Furthermore, the evolution of CIP resistance was more rapid, and resulted in strains with higher MIC values, when the bacteria were biofilm residing than when they were in a planktonic suspension. These results may indicate that extensive clinical AM treatment of biofilm-residing bacteria may not only fail to eradicate the infection but also pose an increased risk of AMR development.

QUIRMIA-A Phenotype-Based Algorithm for the Inference of Quinolone Resistance Mechanisms in Escherichia coli.

Quinolone resistance in Escherichia coli occurs mainly as a result of mutations in the quinolone-resistance-determining regions of gyrA and parC, which encode the drugs' primary targets. Mutational alterations affecting drug permeability or efflux as well as plasmid-based resistance mechanisms can also contribute to resistance, albeit to a lesser extent. Simplifying and generalizing complex evolutionary trajectories, low-level resistance towards fluoroquinolones arises from a single mutation in gyrA, while clinical high-level resistance is associated with two mutations in gyrA plus one mutation in parC. Both low- and high-level resistance can be detected phenotypically using nalidixic acid and fluoroquinolones such as ciprofloxacin, respectively. The aim of this study was to develop a decision tree based on disc diffusion data and to define epidemiological cut-offs to infer resistance mechanisms and to predict clinical resistance in E. coli. This diagnostic algorithm should provide a coherent genotype/phenotype classification, which separates the wildtype from any non-wildtype and further differentiates within the non-wildtype. Phenotypic susceptibility of 553 clinical E. coli isolates towards nalidixic acid, ciprofloxacin, norfloxacin and levofloxacin was determined by disc diffusion, and the genomes were sequenced. Based on epidemiological cut-offs, we developed a QUInolone Resistance Mechanisms Inference Algorithm (QUIRMIA) to infer the underlying resistance mechanisms responsible for the corresponding phenotypes, resulting in the categorization as "susceptible" (wildtype), "low-level resistance" (non-wildtype) and "high-level resistance" (non-wildtype). The congruence of phenotypes and whole genome sequencing (WGS)-derived genotypes was then assigned using QUIRMIA- and EUCAST-based AST interpretation. QUIRMIA-based inference of resistance mechanisms and sequencing data were highly congruent (542/553, 98%). In contrast, EUCAST-based classification with its binary classification into "susceptible" and "resistant" isolates failed to recognize and properly categorize low-level resistant isolates. QUIRMIA provides a coherent genotype/phenotype categorization and may be integrated in the EUCAST expert rule set, thereby enabling reliable detection of low-level resistant isolates, which may help to better predict outcome and to prevent the emergence of clinical resistance.

Fluoroquinolone-resistance mechanisms and molecular epidemiology of ciprofloxacin-resistant Klebsiella pneumoniae isolates in Iran.

Klebsiella pneumoniae is an important cause of nosocomial infections and displays increasing resistance to fluoroquinolones (FQ). This study surveyed the mechanisms of FQ resistance and molecular typing of K. pneumoniae isolates from intensive care units patients in Tehran, Iran. A total of 48 ciprofloxacin (CIP) resistant K. pneumoniae isolates from urine samples were included in this study. Broth microdilution assays revealed high-level CIP resistance (MIC > 32μg/mL) in 31.25% of the isolates. Plasmid-mediated quinolone resistance genes were detected in 41 (85.4%) isolates. Among which, qnrS (41.67%) was the most prevalent followed by qnrD (35.42%), qnrB (27.1%), qnrA (25%), qepA (22.9%), aac(6')-Ib-cr (20.83%), and qnrC (6.25%). Target site mutations (gyrA and parC) were assessed using PCR and sequencing on all isolates. A single mutation in gyrA (S83I) was found in 13 (27.1%) isolates and two isolates harbored six simultaneous mutations. Fourteen isolates (29.2%) had mutations in parC and S129A and A141V mutations were the most prevalent. Real time PCR showed an increase in the expression level of acrB and oqxB efflux genes in 68.75 and 29.16% isolates, respectively. Enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed 14 genotypes and 11 of them were classified by multilocus sequence typing (MLST) into 11 different sequence types belonging to seven clonal complexes and two singletons, most of them have not been reported in Iran yet. We are concerned about the spread of these clones throughout our country. Most FQ resistance mechanisms were detected among our isolates. However, target site mutation had the greatest effect on CIP resistance among our isolates.

The genomic characterization of Salmonella Paratyphi A from an outbreak of enteric fever in Vadodara, India.

Salmonella enterica Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A) are the causative agents of enteric fever, a systemic human disease with a burden of 300 000 cases per year in India. The majority of enteric fever cases are associated with S. Typhi, resulting in a paucity of data regarding S. Paratyphi A, specifically with respect to genomic surveillance and antimicrobial resistance (AMR). Here, we exploited whole-genome sequencing (WGS) to identify S. Paratyphi A genotypes and AMR determinants associated with an outbreak of S. Paratyphi A in Vadodara, India, from December 2018 to December 2019. In total 117 S. Paratyphi A were isolated and genome sequenced, most were genotype 2.4.2 (72.6 % of all cases), which is the globally dominant genotype. The remainder were genotype 2.3 (25.6 %), while only two isolates belonged to genotype 2.4.1. A single base-pair mutation in gyrA, associated with reduced susceptibility to fluoroquinolones, was present in all of the outbreak isolates; with 74.35 % of isolates having a S83F substitution and the remainder having an S83Y substitution. Our surveillance study suggests that S. Paratyphi A is an emergent pathogen in South Asia, which may become increasingly relevant with the introduction of Vi conjugate vaccines.

Molecular Characterization of Resistance to Second-Line Anti-Mycobacterial Drugs among Clinical Isolates of Multidrug-Resistant Mycobacterium tuberculosis

The emergence of multidrug resistance and extensively drug-resistant tuberculosis is a serious public health crisis. Using rapid and inexpensive molecular methods such as HRM assay in the detection of second-line drugs resistance in M. tuberculosis would be helpful in the treatment and control of XDR tuberculosis cases. MDR-TB isolates were collected from Iranian tuberculosis laboratories. Drug susceptibility test performed via the indirect proportion method utilizing LJ Medium. Susceptibility to ciprofloxacin, ofloxacin, amikacin, kanamycin, and capreomycin, as second-line anti-tuberculosis agents were assessed. Single point mutations in gyrA, rrs and eis genes were detected via HRM assay and DNA sequencing. A DST test was performed for 56 MDR isolates and at least 27 (48.2%) isolates were resistant to CIP or OFL. Also, 14 (25%), 12 (21.4%), and 15 (26.7%) isolates were resistant to capreomycin, amikacin, and kanamycin, respectively. D94G, A90V, and G88C mutations were the most frequent mutations in gyrA gene. Also, A1401G mutation was detected more than the other mutations in rrs gene. The frequency of CIP/OFL and AMK/CAP/KAN-resistant TB is considerable among Iranian tuberculosis cases. HRM assay is a rapid and inexpensive test and can detect important mutation-based drug resistance in MDR-TB and XDR-TB isolates.

Comparison of genome-derived and phenotypic antimicrobial resistance profiles of Shigella species isolated from patients with symptoms of gastrointestinal disease in England, 2015-2020

Increasing antimicrobial resistance (AMR) in Shigellaspecies is a global public health problem. We compared genotypic and phenotypic methods for the detection of AMR in Shigellaspecies to evaluate the use of genome data for surveillance and monitoring of emerging AMR. Whole genome sequencing (WGS) data from 388 isolates of all four Shigella species were analysed for the presence/absence of specific AMR determinants and selected accordingly. Phenotypic antimicrobial susceptibility testing was performed using in-agar dilution on all viable and pure isolates (n=358). The genotypic and phenotypic AMR profiles were then compared. There were 335 (93.6%) isolates resistant to at least one antimicrobial and 222 (62%) isolates were multi-drug resistant, of which the majority (77%) were associated with foreign travel. Out of a possible 2864 isolate/antimicrobial class combinations, we identified 119 unexpected results, giving an overall concordance of 96.8% between the two methods. There were 54 samples that had an AMR determinant expected to confer resistance that were phenotypically susceptible, of which 31/54 (57.4%) were associated with tetracycline resistance and trimethoprim-sulfamethoxazole resistance. There were 65 that were phenotypically resistant to a specific antimicrobial class, but no AMR determinant was detected, of which 32/65 (49.2%) were associated with isolates harbouring a single gyrA mutation and exhibiting an unexpectedly high minimum inhibitory concentration (MIC) to ciprofloxacin. Although comparisons between both methods showed good correlation between the genotypic and phenotypic AMR profiles, phenotypic monitoring is required to identify novel AMR mechanisms and to update reference database used for WGS analysis.

Whole Genome Sequencing, Antibiotic Resistance, and Epidemiology Features of Nontyphoidal Salmonella Isolated From Diarrheic Children: Evidence From North China.

Nontyphoidal Salmonella (NTS) in children remains a growing burden on public health and often causes children to be hospitalized with diarrheic symptoms. In this work, 260 strains of human Salmonella isolated from Jilin, China were characterized by serotypes and antimicrobial resistance using whole genome sequencing (WGS). The most prevalent serotype was Salmonella enteritidis (47.3%), followed by S. I 4,[5],12:i:- (33.1%), and Salmonella Typhimurium (7.3%). Furthermore, the consistency between resistance phenotype and genotype was confirmed. Similarly, strains harbored blaTEM−1B and tetA genes were detected, which verified the level of resistant phenotype in β-lactams and tetracyclines. The presence of a single mutation in parC, gyrA, and qnrS1 genes corresponding to quinolones was also observed. In our work, multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) were found to have a high resolution to molecular traceability, and the combination of both was conducive to practical application in an actual situation. Taking all of this into account, we suggested that the comprehensive surveillance of Salmonella infection in children should be carried out to monitor antimicrobial-resistant trends from various sources and to alert on outbreaks of foodborne diseases to protect public health.

Molecular characterisation of antimicrobial resistance determinants and class 1 integrons of Salmonella enterica subsp. enterica serotype Enteritidis strains from retail food in China

Salmonella is a genus responsible for a majority of foodborne disease outbreaks affecting public health worldwide, and Salmonella enterica subsp. enterica serotype Enteritidis (S. Enteritidis) is the most common pathogen that presents alarming rates of antimicrobial resistance. The aim of this study was to analyse the antimicrobial resistance of 124 S. Enteritidis strains collected from retail foods in 39 cities across China and to conduct a comprehensive molecular characterisation of their antimicrobial resistance genes (ARGs) and class 1 integrons through PCR amplification, antibiotic susceptibility testing, and conjugation assays. The strains exhibited a multidrug resistance rate as high as 68.5% and were frequently resistant to nalidixic acid (97.6%) and ampicillin (74.2%). Correspondingly, the analysis of quinolone genes revealed that 97.5% of the strains harboured single mutations in gyrA; some possessed the plasmid-mediated quinolone resistance genes qnrS (0.8%), oqxAB (2.4%), as well as the blaTEM-1 (67.7%); and the extended spectrum beta-lactamase (ESBL) genes blaCTX-M-55 were detected in 2.4% of the strains. Class 1 integrons were observed in 11.3% (14/124) of the strains and integrated three different gene cassettes, namely, aadA22 (1/14), dfrA1-aadA1 (5/14), and dfrA17-aadA5 (8/14). Conjugation experiments and antimicrobial susceptibility testing showed that the integrons including antimicrobial resistance genes could be transferred to the recipient strain via mobile plasmids, rendering the recipient strain resistant. Both multilocus variable-number tandem-repeat analysis (MLVA) and core genome multilocus sequence typing (cgMLST) show a better discrimination in this study, hence a combination of MLVA and cgMLST analyses would improve the accuracy and cost-effectiveness. This study provides a systematic and comprehensive analysis of the antimicrobial resistance profiles of S. Enteritidis strains isolated from retail food in China. The present data will help to gain insight into the public health impact of S. Enteritidis and guide the development of control strategies.

Identification of two novel type II topoisomerase mutations in Enterococcus spp. isolated from a hospital in China

Type II topoisomerases, including DNA gyrase (GyrA) and topoisomerase IV (ParC), contribute to fluoroquinolone resistance in Enterococcus spp. This study investigated the mutational status of the quinolone resistance-determining regions (QRDRs) of GyrA and ParC in the clinical isolates of enterococci from a hospital in Baotou, China. We analyzed 110 enterococcal isolates, including 57 Enterococcus faecalis and 53 Enterococcus faecalis faecium. The resistance rates of E. faecalis and E. faecium to ciprofloxacin were 63.16% and 84.91%, respectively. We found that 32 samples of E. faecalis and 42 of E. faecium had single or combined mutations in gyrA and/or parC, which were all resistant to ciprofloxacin. Only two ciprofloxacin-resistant E. faecalis isolates had no mutation. No mutations in gyrA and parC genes in all ciprofloxacinsusceptible isolates were found. Ciprofloxacin minimal inhibitory concentrations (MICs) in the mutation group were significantly higher than those of the non-mutation group, indicating that mutations in the QRDRs of gyrA and parC were correlated with MIC elevation. Two novel substitutions (GyrA Ser83Phe and ParC Ser80Leu) of E. faecalis were identified herein. Three-dimensional modeling revealed that these novel amino acid substitutions could disrupt the water/metal-ion bridge and decrease the interaction between the enzymes and ciprofloxacin. The data showed a diversity of mutation types in QRDRs of type II topoisomerases whose association with fluoroquinolone resistance in clinical isolates of enterococci warrants further investigation.

English

Enteric fever is a systemic disease classified into typhoid and paratyphoid fever. Fluoroquinolones and third generation cephalosporins are usually the drugs of choice in the management of Salmonella infections. Previous reports have indicated common occurrence of multi-drug resistance (MDR) including resistance to β-lactams and fluoroquinolones. However, there is paucity of information on the genetic determinants of resistance to β-lactam and fluoroquinolones from S. enterica in Southeast Nigeria. Salmonella enterica serovars were identified and screened (25 each from unrelated patients from the four hospitals). Resistant isolates were screened for ESBL phenotypically. Genomic and plasmid DNA were extracted by boiling and alkaline lysis, respectively. PCR amplification of blaTEM, blaSHV and blaCTX-M, among the ESBL positive isolates and sequencing of the Quinolone Resistance Determining Regions (QRDR) on fluoroquinolone resistant isolates were determined. Of the 100 isolates, thirty six of the MDR isolates produced ESBL phenotypically, of which 13 were blaCTX-M positive. DNA sequencing revealed single point mutations in gyrA at amino acid positions Asp-87-Gly, Asp-87-Asn and Ser-83-Tyr in 55 (68.8%), and double mutation in parC at positions Asp-87-Gly in 14 (17.5%). Mutations in gyrA, parC genes, and chromosomal blaCTX-M were responsible for the fluoroquinolones and cephalosporins resistance, respectively in some of the Salmonella enterica from Southeast Nigeria. QRDR of the gyrA gene of the isolates sequenced showed reduced susceptibility to some fluoroquinolone. The taxonomic and neighbouring trees of similar species causing infection worldwide were identified, and hence, alleviates the fear of easy spreading of quinolone and cephalosporin resistant isolates. Key words: Quinolone Resistance Determining Regions (QRDR), ESBL, S.enterica, mutation.  

Antibacterial activity of novel dual bacterial DNA type II topoisomerase inhibitors.

In this study, a drug discovery programme that sought to identify novel dual bacterial topoisomerase II inhibitors (NBTIs) led to the selection of six optimized compounds. In enzymatic assays, the molecules showed equivalent dual-targeting activity against the DNA gyrase and topoisomerase IV enzymes of Staphylococcus aureus and Escherichia coli. Consistently, the compounds demonstrated potent activity in susceptibility tests against various Gram-positive and Gram-negative reference species, including ciprofloxacin-resistant strains. The activity of the compounds against clinical multidrug-resistant isolates of S. aureus, Clostridium difficile, Acinetobacter baumannii, Neisseria gonorrhoeae, E. coli and vancomycin-resistant Enterococcus spp. was also confirmed. Two compounds (1 and 2) were tested in time-kill and post-antibiotic effect (PAE) assays. Compound 1 was bactericidal against all tested reference strains and showed higher activity than ciprofloxacin, and compound 2 showed a prolonged PAE, even against the ciprofloxacin-resistant S. aureus BAA-1720 strain. Spontaneous development of resistance to both compounds was selected for in S. aureus at frequencies comparable to those obtained for quinolones and other NBTIs. S. aureus BAA-1720 mutants resistant to compounds 1 and 2 had single point mutations in gyrA or gyrB outside of the quinolone resistance-determining region (QRDR), confirming the distinct site of action of these NBTIs compared to that of quinolones. Overall, the very good antibacterial activity of the compounds and their optimizable in vitro safety and physicochemical profile may have relevant implications for the development of new broad-spectrum antibiotics.

Prevalence and Characteristics of Quinolone Resistance in Salmonella Isolated from Retail Foods in Lanzhou, China

The aim of this study was to determinate the prevalence of Salmonella in retail foods and its resistance to quinolones in retail foods in Lanzhou, People's Republic of China. In this work, 2,182 food samples, collected from March 2015 to December 2018, were analyzed to detect Salmonella and then analyzed for serotype distribution, quinolone resistance, and quinolone-resistant gene detection. The findings demonstrate that the overall prevalence of Salmonella in these food categories was low. A total of 41 (1.9%) of 2,182 food samples were found to be positive for Salmonella. Ten distinct serovars were identified, and Salmonella Derby, Salmonella Anatum, and Salmonella Enteritidis were the most prevalent serovars. According to the broth microdilution test, the resistance percentages were 90.2% to nalidixic acid, 39.0% to enrofloxacin, 41.5% to ciprofloxacin, 29.3% to ofloxacin, and 26.8% to levofloxacin. Among the quinolone-resistant isolates, 12 strains had a single mutation in gyrA at codon 83 (Ser→Phe) or codon 87 (Asp→Asn or Asp→Gly). Five isolates had one parC mutation (Ser80→Arg) and one or two gyrA hot spot mutations. qnr genes were found in seven isolates (five qnrB and two qnrD), and the aac(6')-Ib gene in seven isolates. Two isolates carry both qnrB and aac(6')-Ib-cr genes. Based on these results, a low prevalence of Salmonella contamination in retail foods was found, but it might play a potential risk factor in the spread of quinolone-resistant Salmonella strains in the Lanzhou region.

Genetic Characterization of Fluoroquinolone Resistance in Salmonella enterica Serovar Gallinarum Isolates from Chicken in Korea.

Salmonella enterica serovar Gallinarum is a nonmotile host-adapted Salmonella that causes fowl typhoid (FT), and an outbreak of FT is characterized by anorexia, greenish-yellow diarrhea, paleness, and sudden death with high mortality in poultry. To control and treat FT in commercial chickens, fluoroquinolones are widely used in Korea. This study aimed to investigate the genetic characteristics of fluoroquinolone-resistant Salmonella Gallinarum isolates from 2014-18 from chicken in Korea. A total of 35 ciprofloxacin (CIP)-resistant Salmonella Gallinarum was tested, and 22 (62.9%) isolates were observed to have multidrug resistance. All isolates had a mutation at the Ser83 or Asp87 codon in the gyrA gene, whereas three isolates had only double mutations at Ser83 → Phe and Asp87 → Asn or Ser83 → Phe and Asp87 → Gly. Minimum inhibitory concentrations of isolates with double mutations were relatively higher (≥8 mg/L for CIP and ≥16 mg/L for enrofloxacin) than those of other isolates with a single mutation in gyrA. Among 35 CIP-resistant Salmonella Gallinarum, plasmid-mediated quinolone resistance genes were detected in six (17.1%) isolates, and qnrB and qnrS were detected in four and two isolates, respectively. In the distribution of antimicrobial resistance genes in 35 CIP-resistant Salmonella Gallinarum, ant(2″)-I (54.3%) was the most prevalent gene, followed by TEM-1 (14.3%), sul1 (11.4%), and cmlA (5.7%). Fifteen (42.9%) of the 35 CIP-resistant Salmonella Gallinarum also carried class 1 integrons, which showed five types of resistance gene cassettes: aadA2 (7 isolates), aadA2 + dfrA12 (5 isolates), and aadA1 + aad A2 (3 isolates). Among plasmid replicons, 23 isolates (65.7%) carried five different plasmid replicons: Frep (9 isolates), FIB (7 isolates), FIIA (6 isolates), B/O (4 isolates), and I1 (3 isolates). These results suggest that continued monitoring of fluoroquinolone resistance is necessary to preserve the effectiveness of fluoroquinolones in poultry and to surveil the transmission to humans through the food chain.

Emergence of phylogenetically diverse and fluoroquinolone resistant Salmonella Enteritidis as a cause of invasive nontyphoidal Salmonella disease in Ghana.

BackgroundSalmonella enterica serovar Enteritidis is a cause of both poultry- and egg-associated enterocolitis globally and bloodstream-invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa (sSA). Distinct, multi-drug resistant genotypes associated with iNTS disease in sSA have recently been described, often requiring treatment with fluoroquinolone antibiotics. In industrialised countries, antimicrobial use in poultry production has led to frequent fluoroquinolone resistance amongst globally prevalent enterocolitis-associated lineages.Methodology/Principal findingsTwenty seven S. Enteritidis isolates from patients with iNTS disease and two poultry isolates, collected between 2007 and 2015 in the Ashanti region of Ghana, were whole-genome sequenced. These isolates, notable for a high rate of diminished ciprofloxacin susceptibility (DCS), were placed in the phyletic context of 1,067 sequences from the Public Health England (PHE) S. Enteritidis genome database to understand whether DCS was associated with African or globally-circulating clades of S. Enteritidis. Analysis showed four of the major S. Enteritidis clades were represented, two global and two African. All thirteen DCS isolates, containing a single gyrA mutation at codon 87, belonged to a global PT4-like clade responsible for epidemics of poultry-associated enterocolitis. Apart from two DCS isolates, which clustered with PHE isolates associated with travel to Spain and Brazil, the remaining DCS isolates, including one poultry isolate, belonged to two monophyletic clusters in which gyrA 87 mutations appear to have developed within the region.Conclusions/SignificanceExtensive phylogenetic diversity is evident amongst iNTS disease-associated S. Enteritidis in Ghana. Antimicrobial resistance profiles differed by clade, highlighting the challenges of devising empirical sepsis guidelines. The detection of fluoroquinolone resistance in phyletically-related poultry and human isolates is of major concern and surveillance and control measures within the region’s burgeoning poultry industry are required to protect a human population at high risk of iNTS disease.

Changes in Outer Membrane Proteins of Benzalkonium Chloride Adapted Pseudomonas Aeruginosa and Mutations in GyrA and ParC Genes

AbstractBackground: Benzalkonium Chloride (BC) is widely used in hospitals, industry and cosmetics. Adaptation of Pseu-domonas aeruginosa to BC was increased. This adaptation may lead to the emergence of cross-resistance to other disin-fectants and antibiotics. Little attention has been focused on the resistant mechanisms.Aim of Study: Examination of mutations in the Quinolone-resistance-Determining Region (QRDR) of GyrA and ParC genes and alterations of Outer-Membrane Proteins (OMPs) in Benzalkonium Chloride (BC)-adapted Pseudomonas aeru-ginosa isolates.Material and Methods: GyrA and ParC genes of the BC-adapted P. aeruginosa isolate and the wild-type strain ATCC 15442 were amplified by Polymerase-Chain-Reaction (PCR) and sequenced. OMPs of these isolates were also analyzed by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophore-sis (SDS-PAGE) in the presence and absence of permeabilizer disodium Ethylenediamine-Tetraacetate (EDTA) to investigate the variations.Results: The results manifested a single mutation in both GyrA (Thr-83-Ile) and ParC (Ser-87-Leu). In SDS-PAGE, one band with molecular weight of 52.23kDa was detected in all samples. Seven bands with molecular weights of 71.44, 38.48, 36.01, 33.03, 26.17, 21.14 and 6.35kDa were stable in both ATCC 15442 and treated isolate. Also, seven bands with molecular weights of 120.83, 90.89, 79.72, 44.58, 28.91, 16.12 and 12.49kDa were newly induced in the treated isolate.Conclusions: These results revealed that mutations in GyrA and ParC genes, and alteration of OMPs contributed to the adaptation of P. aeruginosa to BC, co-operating with efflux pump over-expression.

Genetic mutations in the quinolone resistance-determining region are related to changes in the epidemiological profile of methicillin-resistant Staphylococcus aureus isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important causative agent of nosocomial infections. Mutations in the quinolone resistance-determining regions (QRDRs) of the gyr and par genes have been described. This study aimed to characterise phenotypic and genotypic fluoroquinolone resistance in 69 MRSA isolates of different clonal lineages from hospitals in Rio de Janeiro, Brazil. QRDR mutations in the gyrA, gyrB, parC and parE genes were detected by DNA sequencing. Minimum inhibitory concentrations (MICs) for ciprofloxacin and moxifloxacin were determined by broth microdilution. The occurrence of associations between mutations and MICs among the different clonal lineages of MRSA isolates was then verified. Most isolates from the USA400/ST1/SCCmec IV lineage, but mainly USA100/ST5/SCCmec II isolates, which have been more recently found in Rio de Janeiro hospitals, showed different patterns of mutations, including double mutation in the QRDR of parC (Ser-80⿿⿿⿿Tyr and Glu-84⿿⿿⿿Lys/Gly) and/or gyrA (Ser-84⿿⿿⿿Leu and/or Glu-88⿿⿿⿿Lys) associated with higher moxifloxacin and ciprofloxacin MICs (MIC90, ⿥8⿿mg/L and 256⿿mg/L, respectively). On the other hand, all USA800/ST5/SCCmec IV and the BEC/ST239/SCCmec III isolates, which have disappeared from hospitals, showed single mutations in parC (Ser-80⿿⿿⿿Phe) and gyrA (Ser-84⿿⿿⿿Leu or Glu-88⿿⿿⿿Gly) and lower fluoroquinolones MICs (MIC90, ⿥2⿿mg/L and ⿥16⿿mg/L). This study highlights an increase in the number and types of mutations in the QRDRs ofgyrA and parC associated with high fluoroquinolones MICs that may be related to changes in the epidemiological profile of MRSA isolates from hospitals in Rio de Janeiro.

Molecular surveillance of antimicrobial resistance and transmission pattern of Mycobacterium leprae in Chinese leprosy patients

ABSTRACT Reports on antimicrobial resistance (AMR) of Mycobacterium leprae, relationship with bacteriological index (BI), and transmission in China are limited. We investigated the emergence of AMR mutations, the relationship between BI and AMR in complete, moderate and lack of BI decline cases, and molecular epidemiological features of AMR cases by enrolling 290 leprosy cases from four endemic provinces. Seven (2.41%), one (0.34%), five (1.72%), one (0.34%), and one (0.34%) strains had single mutations in folP1, rpoC, gyrA, gyrB, and 23S rRNA, respectively. Double mutations in folP1 and gyrA, rpoB and gyrA, and gyrA and 23S rRNA were observed in one (0.34%) strain each. Mutated strains occurred in three out of 81 (95% CI−0.005-0.079, p = 0.083) cases with complete BI decline, in seven out of 103 (95% CI 0.018-0.117, p = 0.008) cases with moderate BI decline, and in four out of 34 (95% CI 0.003-0.231, p = 0.044) cases with lack of BI decline. Most of these mutated strains were geographically separated and diverged genotypically. AMR mutations may not be the main cause of the lack of BI decline. The low transmission of AMR strains at the county level indicates an ongoing transmission at close contact levels.

Molecular typing and profiling of topoisomerase mutations causing resistance to ciprofloxacin and levofloxacin in Elizabethkingia species.

ObjectivesSeveral Elizabethkingia species often exhibit extensive antibiotic resistance, causing infections associated with severe morbidity and high mortality rates worldwide. In this study, we determined fluoroquinolone susceptibility profiles of clinical Elizabethkingia spp. isolates and investigated the resistance mechanisms.MethodsIn 2017–2018, 131 Elizabethkingia spp. isolates were recovered from specimens collected at tertiary care centers in northern Taiwan. Initial species identification using the Vitek MS system and subsequent verification by 16S rRNA sequencing confirmed the presence of Elizabethkingia anophelis (n = 111), E. miricola (n = 11), and E. meningoseptica (n = 9). Fluoroquinolone susceptibility was determined using the microbroth dilution method, and fluoroquinolone resistance genes were analyzed by sequencing.ResultsAmong Elizabethkingia spp. isolates, 91% and 77% were resistant to ciprofloxacin and levofloxacin, respectively. The most prevalent alterations were two single mutations in GyrA, Ser83Ile, and Ser83Arg, detected in 76% of the isolates exhibiting fluoroquinolone MIC between 8 and 128 μg/ml. Another GyrA single mutation, Asp87Asn, was identified in two quinolone-resistant E. miricola strains. None of the isolates had alterations in GyrB, ParC, or ParE. We developed a high-resolution melting assay for rapid identification of the prevalent gyrA gene mutations. The genetic relationship between the isolates was evaluated by random amplified polymorphic DNA PCR that yielded diverse pulsotypes, indicating the absence of any temporal or spatial overlap among the patients during hospitalization.ConclusionOur analysis of fluoroquinolone-resistant Elizabethkingia spp. isolates provides information for further research on the variations of the resistance mechanism and potential clinical guidance for infection management.

Relationship between danofloxacin PK/PD parameters and emergence and mechanism of resistance of Mycoplasma gallisepticum in In Vitro model.

Mycoplasma gallisepticum is a serious pathogen for poultry that causes chronic respiratory disease in chickens. Increased embryonic mortality, as well as reduced weight gain and egg production have been found in infected chickens, which can lead to considerable economic losses in poultry production. Increased antibiotic resistance compromises the use of tetracyclines, macrolides and quinolones in the farm environment. In the present study, danofloxacin concentrations were simulated below the MIC99, between the MIC99 and MPC (the mutant prevention concentration), and above the MPC in an in vitro dynamic model against M. gallisepticum. The relationship between the simulated danofloxacin pharmacokinetics, pharmacodynamics (PK/PD) parameters and development of resistance for M. gallisepticum was explored based on the available data obtained from various dosing regimens in the in vitro model. Danofloxacin concentration, counts of viable cell and susceptibility were determined during the experiment. The mutations in gyrA, gyrB, parC and parE as well as efflux pumps were examined. The MIC of danofloxacin against M. gallisepticum was increased when drug concentrations were between the lower and upper boundaries of the mutant selection window. The upper boundary of the selection window in vitro was estimated as a Cmax/MPC value of 1. The lower boundary was estimated as Cmax/MPC value of 0.05. Both in terms of the MIC and resistance frequency, M. gallisepticum resistance was developed when danofloxacin concentrations fell inside the mutant selection window (ratios of Cmax to MPC between 0.05 and 1). The single mutation in gyrA (Ser-83→Arg) was found in all mutants, while double mutations in gyrA and parC (Ala-64→Ser) were observed only in the mutant with the highest MIC. In addition, no change of susceptibility in the mutants was observed in the presence of reserpine and carbonyl cyanide 3-chlorophenylhydrazone (CCCP). This suggested that ATP-binding cassette superfamily (ABC transporter) and major facilitator superfamily (MFS transporter) did not play a role in danofloxacin efflux.

Molecular Study of Quinolone Resistance Determining Regions of gyrA Gene and parC Genes in Clinical Isolates of Acintobacter baumannii Resistant to Fluoroquinolone.

Introduction: Acinetobacterb aumannii (A. baumannii) is an important pathogen in health care associated infections. Quinolone resistance has emerged in this pathogen.Aims & Objectives:The aim of the present study was to determine the presence of mutations of gyrA gene and parC genes by Restriction Fragment Length Polymorphism Polymerase Chain Reaction (RFLP-PCR) among clinical isolates of A. baumanii.Materials and Methods:The study was carried out on 140 clinical isolates of A. baumannii. The isolates were subjected to molecular study of mutations of gyrA gene and parC genes by RFLP–PCR beside determination of Minimal Inhibitory Concentration (MIC) by macro dilution tube method.Results:The isolates of A. baumannii were resistant to ciprofloxacine and levofloxacin at MIC >4 µg/ml. The most isolates had MIC >128 µg/ml (42.3%). All resistant strains to ciprofloxacin of A. baumannii had mutations in gyrA and parC. The most frequent mutations were combined mutations in both genes (85.5%) and 5% had single mutation either in gyrA or parC. The most frequently combined mutations were associated with MIC >128 µg/ml (42.3%).Conclusion:From this study we can conclude that resistance to ciprofloxacin was common in clinical isolates of A. baumannii. The most frequent mutations were present in gyrA and parC. However, mutations in parC alone were not uncommon. Further large scale studies are required to elucidate the resistance pattern of A. baumannii and its molecular mechanisms.